1. Field of the Invention
The present invention relates generally to detectors based on binding events and, more specifically, to fluidized bed detectors for continuous, ultra-sensitive detection of biological and chemical materials.
2. Description of the Prior Art
There is a continued need for sensors for materials such as chemicals, bacterial toxins, and viruses at small concentrations in a large volume of media, where media may be defined as a solvent, such as water, a solid, or a gas or a mixture of media such as food which contains all three states. Some substances that affect biological function are known, so that specific detectors are possible, and some are unknown, so only living organisms can be the sentinel detector. Much effort has been and is being spent on assays for these materials that employ living cells, such as bacteria, mammalian cells, or small organisms as the detection element. The classic example is the use of canaries in mines to detect harmful gases. Because the canaries have a smaller body mass than humans and a higher respiratory rate, they tend to succumb first if harmful gases are present. Live bioassays have a number of issues that limit their use. (1) The living organism must be kept alive. This requires some care and feeding even when the “sensor” is not being actively used. (2) Live bioassays that use complex organisms (such as canaries) may respond to other factors that do not cause appreciable concern for humans, such as temperature and transportation stress. (3) Interpretation of the response of the complex organism may be difficult in varying environments. For example, why did the canary die? Was it a toxic substance in the air, heat stress, or an avian virus that only affects canaries but not humans or other species? (4) For cell-based live assays, the cells also must be kept alive and more importantly some transduction mechanism must be engineered into the cells to identify the threat. For example, the transduction mechanism may be the production of green fluorescent protein or firefly luciferase in response to certain substances. In these cases, the cells would be monitored for an increase in fluorescence or light emission upon addition of suitable substrates. Although such a cell-based, live bioassay can be useful, each organism must be engineered to respond to a unique substance, which can be a daunting task if the agent in question does not have a known signaling pathway.
If the agent in question is known, specific detectors are possible. They may be either based on immunoassays, where the antibodies or other specific binding molecules such as peptides can act as the recognition element, or nucleic acid detectors where specific sequences of DNA or RNA are sought. The major issue with many specific detectors is that the volume of liquid or air testable is quite small so that a preconcentration step is required. Without this preconcentration step, the likelihood of detection of a target species decreases with the concentration and the volume of material needed for the test (see FIG. 1). Because of this preconcentration step, the testing technology is often a grab-and-sample type of test, i.e., the preconcentrator is run for a given amount of time, a sample of the concentrate is taken, and the sample tested. This process can be repeated on a periodic time scale but as most tests are single-use tests where consumption of reagents can be substantial so that the frequency of testing is often limited. Most immunoassays are single use; an example being the lateral-flow immunoassays used in home-pregnancy testing. An exception is displacement assays such as the flow-immunoassay described in Kusterbeck et al., “A continuous-flow immunoassay for rapid and sensitive detection of small molecules,” Journal Of Immunological Methods, 135, 191-197 (1990), the entire contents of which are incorporated herein by reference. Displacement assays, although proposed here as well, are not as sensitive as completion or sandwich immunoassays due to kinetics and antibody affinity. For high sensitivity, the antibody binding constant must be as high as possible. However, tight binding means slow release so that the displacement with a test antigen either is slow or requires high levels of antigen.
U.S. Patent Application 20040009529 by Weimer et al. (Jan. 15, 2004), the entire contents of which are hereby incorporated by reference, describes a process for the capture of antigens on beads in a flow-though module. This is not a continuous assay as the captured antigens must be detected, (for example with an enzyme—linked antibody) in a grab and sample mode. The particles are not released upon binding to the antigen. This system is similar to the BEADS (Biodetection Enabling Analyte Delivery System) sample preparation technology described in the next paragraph.
U.S. Pat. No. 6,506,584 to Chandler et al. (Jan. 14, 2003), U.S. Pat. No. 6,159,378 to Holman et al. (Dec. 12, 2000), and U.S. Pat. No. 6,136,197 to Egorov et al. (Oct. 24, 2000), the entire contents of each are hereby incorporated by reference, describe PNNL's BEADS (Biodetection Enabling Analyte Delivery System) sample preparation technology. Although claimed as a fluidized bed for rapid kinetics, PNNL does not use a force field to retain the particles but rather a complex capture and release system for the particles to maintain them in a fluidized state. Like the Weimer application, the BEADS system is more a grab and sample system. Furthermore, in the way that the particles are retained (like a simple screen), the BEADS system cannot handle complex media, such as food, as it will quickly plug.
U.S. Pat. No. 6,153,113 to Goodrich et al. (Nov. 28, 2000), the entire contents of which are hereby incorporated by reference, uses a continuous centrifuge and binding particles. However, it is not a detector and it does not release the particles upon binding. Rather the particles only act as a capture medium to remove a selective blood component from the incoming fluid with purification of the fluid (blood in this case) as the goal.
U.S. Pat. No. 4,939,087 to Van Wie et al. (Jul. 3, 1990), the entire contents of which are hereby incorporated by reference, describes the use of a centrifugal reactor for culturing cells and harvesting high-valued proteins in a continuous manner. This patent shows that a centrifugal reactor can maintain living cells on a long-term basis. However, it is not used as a detector.